Fusion Expression Of Somatostatin With Xylanases And Characterization Of The Recombinant Proteins

Somatostatin(SS)inhibits the release of growth hormone and gastric motility,gastric acid secretion,nutrient absorption and hinders feed conversion and animal growth.In animal production practice,if SS is used to actively immunize animals to produce specific antibody against SS and neutralizing it,the negative effect of SS can be prevented.The commonly used immunization methods include injection,oral administration and spraying.Among these methods,oral immunization is convenient and the vaccine can be applied directly to the feed.However,most of the previous studies on the use of SS to immunize animals to improve production performance have used DNA vaccines as carriers.The efficient preparation of recombinant functional SS proteins has been rarely seen.Feeding enzymes are common feed additives.Oral intake of feed enzymes by animals allows the enzymes to exert their functions(including degrading substrates,promoting growth and regulating intestinal flora in the gastrointestinal tract).Therefore,feed enzymes have natural superiority when used as fusion carriers of functional small peptides.Among the feed enzymes,xylanase has been successfully applied in the feed of pigs,chickens and even ruminants.Its application can reduce the viscosity of chyme and improve the feed conversion rate.Unlike another commonly used feed enzyme cellulase,the xylanase currently used is often a single kind of enzyme.In addition,the use of Pichia pastoris to produce recombinant xylanase by fermentation is very mature in China.Therefore,in theory fusion expression of xylanase and SS can be used to efficiently produce SS recombinant protein for oral immunization.In this study we tried to express three xylanases from different microbial sources,namely Np Xyl74(from Neocallimastix patriciarum),CbXyn10C(from Caldicellulosiruptor bescii)and BsXyl11A(from Bispora sp.)by fusing them individually with the somatostatin SS-14.When SS-14 was placed at the Cterminus,all three fusion proteins could be successfully expressed heterologously in Pichia pastoris.Fusion expression did not cause large negative impacts on the physicochemical properties of xylanase,only slightly decreasing the optimal temperature and thermal stability of the xylanases.Through Western blot,we found that the fusion proteins Cb Xyn10C-SS and BsXyl11A-SS could both specifically react with rabbit polyclonal antibody against SS,which indicates that SS in the fusion protein still retains its antigenicity.In the Western blot assay,the ability of Np Xyl74-SS to react with the antibody was less significant than the control NpXyl74.Considering that the protein is highly glycosylated when expressed in Pichia pastoris,the modified glycosylation side chain may affect the antibody's recognition of the fusion protein.We immunized BALB/c mice with the three fusion proteins by subcutaneous injection and prepared antiserum.The dot blot experiments indicated that the three sera could all react with the synthesized SS-14 antigen.This indicated that no matter which xylanase was fused with SS,the fusion protein could induce the body to produce specific antibodies.The three fusion proteins were individually fed to BALB/c mice by gavage.The initial observation suggested that there was no significant difference between the experimental groups and the control groups in terms of weight gain.The reason might be due to the short growth period,inappropriate dosage and the need of a proper adjuvent.Finally,we carried out high density fermentation of the recombinant P.pastoris in the fermentor.After 114 h,the xylanase activities of Np Xyl74-SS and CbXyn10C-SS in the fermentation broth were 8564 U/mL and 541 U/mL,respectively,and the maximum total protein concentrations were 3640 mg/L and 2001 mg/L,respectively.For Bs Xyl11A-SS,the xylanase activity in the fermentation broth after 116 h was 5011 U/mL and the highest total protein concentration was 3697 mg/L.In summary,the xylanase-SS fusion protein not only retains the catalytic activity and thermal stability,pH stability of xylanase,but also can stimulate the animal body to produce specific antibodies against SS-14.Through fermentation in a bioreactor,the recombinant fusion proteins can be prepared in a large scale.Therefore,this method is an effective method for preparing functional SS in large quantities.The fusion protein obtained in the experiment is a bifunctional protein.In the future,chickens,pigs,sheep and other animals with longer growth cycles and larger body types can be further tested for growth promotion.The research provides new ideas for expanding the functions of traditional feed enzymes and the upgrading of such feed enzymes.